IMPROVED DETECTION OF POINT MUTATIONS IN THE HUMAN ANDROGEN RECEPTOR GENE BY DENATURING GRADIENT GEL ELECTROPHORESIS OF DNA HETERODUPLEXES UNDER STRINGENT DENATURING CONDITIONS

Abstract

Single nucleoude missense mutations in the human androgen receptor (AR) gene represent the underlying molecular lesion in most cases of complete or partial androgen insensitivity (AIS). Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified DNA fragments containing a 5 40-base pair GC-clamp has been used successfully to detect point mutations in several human genes, including the AR gene in our laboratory (Mol Endo 4:1759 [1990]). The sensitivity for detection of mutations can be further enhanced by the formation of DNA heteroduplexes and by narrowly defining the denaturing gradient formed by formamide and urea (i.e. 100% denaturant = 7M urea/40% formamide) specific for each region of the gene to be analyzed. In the present study, genomic DNA of normal subjects and those with AIS was amplified by PCR using primers specific for each of the eight exons encoding the human AR. Amplified DNA from affected subjects was mixed with that from a normal subject, heat denatured at 95 C for 5 min and then allowed to reanneal at room temperature to form both homo- and hetero-duplexes. The DNA samples were electrophoresed at 60 C on a 6.5% polyaerylamide gel containing a gradient of increasing concentrations of formamide and urea. The presence of ethidium bromide stained DNA species with differing mobilities within a single sample was indicative of heteroduplex formation and the presence of a point mutation which was subsequently confirmed by nucleotide sequence analysis. We detected three new point mutations in the AR gene. In one subject with partial AIS, amino acid residue 913 encoded within exon 8 is mutated [CCC(pro)→TCC(ser); P913S]. In another subject with the partial form of AIS, codon 840 in exon 7 is mutated [CGT(arg→CAT(his); R840H). In one subject with complete AIS, codon 732 in exon 5 is mutated (GAC(asp)→TAC(tyr); D732V]. The use of a 35%-70% rather than the usual 20-80% denaturant gradient was required to detect the mutation in exon 8, whereas the heteroduplex analysis was critical for the detection of the mutation in exon 5. These findings demonstrate the utility of heteroduplex formation and stringent denaturant conditions to improve the detection of some mutations in the AR gene using DGGE. (Supported in part by NIDDK R01-43417 and CNR AI90-01384-04)